Solid-Phase Combinatorial Technology

to Speed-up Nucleoside Synthesis

Haoyun An

1st e-CombiChem ConferenceCutting Edge Technologies in Combinatorial Chemistry

June 14-25, 2004

Integrated Infrastructure to Success

ChemistryCombinatorial Chemistry

Organic Chemistry

Nucleoside Chemistry

Medicinal Chemistry

Crystallography

SBDD

Automation

Compound Control

Data Management

Drug Development

ADMET

Chem-Informatics

Discovery Analytical

High Throughput

LC-MS/Purification

Library Acquisition

Therapeutic AreasVirology

Oncology

Immunology

Drugs

SARHTS

Strategies to Enhance Productivity

▲Traditional Approach:

► 75-100 compounds / year / person

▲ Parallel Solution-Phase Combinatorial:

► 500-800 / year / person (Purification issues)

▲ Parallel Solid-Phase Combinatorial:

► 1000-5000 / year / person (after scaffold development)

▲ Complex / Mixture Libraries:

► 1000-5000 / year (require deconvolution)

Combinatorial & Nucleoside Chemistry

▲ Combinatorial Chemistry

►15 years - peptides, oligos, heterocycles

►>100 companies / labs

▲ Nucleoside Chemistry

► 60 years - natural and unnatural

nucleosides

► >80 universities / companies

Combinatorial Approaches

For Nucleoside Libraries

▲ Challenges -

►Almost nobody made nucleoside libraries by

solid-phase combinatorial approaches

►Difficult to apply combichem approaches to

nucleosides - special structures Difficulty & Low yield

No harsh condition

Protection and de-protection

►We have pioneered this combination to

accelerate combinatorial nucleoside drug

discovery process

Resins for Nucleoside Libraries

Resin:

Wang

MMT-Cl

Merrifield

Cleavage:

50% TFA/DCM

1~1.5% TFA/DCM or

Hexafluoroisopropanol

HF orstrong base

► >100 Resins available for solid-phase reactions

► MMT-Cl Resin is applicable for nucleoside synthesis

N N

NN

N

O

OPGGPO

O

R1

R2

R4R3

Cl

O

X

Cl

OMe

Validation

Validate loaded resins

Validate building blocks

Validate the protocols

Validate reaction conditions

Combinatorial Process

Scaffold/

library

design

Scaffold

synthesis/

feasibility

research

Solid phase

chemistry

Large scale

synthesis of

Resin loaded

with scaffolds

Protocol

validationBuilding block

validation

96-Well

plates

synthesis

Enumeration

plate design

Library Design - Afferent

Reagent selection – ACD

Enumeration - Afferent

Combinatorial Process

96-Well

plates

Dry

samples

Sample

preparation

HTP LC-MS

analysis

Results

analysisRegister

in Database

HTP

purification

60-100%

LC-MS

Pooling

Registration Library - Afferent

HTP purification - Paralex

30-60%

Development of 2’-O-Me-N4-Substituted

Cytidine Scaffold in Solution

NO

OCH3TBDMSO

MMTO

NH

O

O

NO

OCH3TBDMSO

MMTO

N

O

OTPS

NO

OCH3HO

HO

NH

O

O

NO

OCH3HO

MMTO

NH

O

O

NO

OHHO

HO

NH

O

O

Pri

ClSO2

Pri

Pri

TPS-Cl

py / DMAP

MMTCl

Py

TBDMSCl

Py / DMAP

85% 89%

76%

NO

OCH3TBDMSO

MMTO

N

O

NNHN

NNH2N

NO

OCH3HO

HO

N

O

NNHN

Et3N / DMAP, r.t., o/n

80%

1, TBAF, rt, 3 h

2, TFA80%Confirmed by NMR and LC-MS

2’-O-Me-N4-Substituted Cytidine Scaffold

on Solid Support

NO

OCH3TBDMSO

NH

O

O

O

NO

OCH3TBDMSO

N

O

OTPS

O

NO

OCH3HO

HO

NH

O

O

NH

NO

OCH3TBDMSO

N

OO

NH2

MMTCl

NH

NO

OCH3HO

N

OHO

Pri

ClSO2

Pri

Pri

py / DMAP / TEA rt, 24 h

TBDMSCl

Py / DMAP / TEADMF / imidazole rt, 24 h

1, TBAF, rt, 5 h

2, TFA, 10 min

Py / DMAPrt, 24 h

4.5 g (7 mmol)

(1.2 equiv)

DMAP, Hunig's basepy, DMF, rt, o/n

LC-MS: 93%ES MS: confirmed

7.2 g ResinMAS NMR confirmed

TPS-Cl(2 equiv)

exo-2-aminonorbornane

2’-O-Me-N4-Substituted Cytidine Library

14 plates

1244 compds

NO

OCH3TBDMSO

NH

O

O

O

NO

OCH3TBDMSO

N

O

O

O

NO

OCH3HO

HO

NH

O

O

MMTCl

R1

N

NO

OCH3HO

N

OHO

Pri

ClSO2

Pri

Pri

py / DMAP / TEA rt, 24 h

TBDMSClPy / DMAP

TEA/DMF imidazole rt, 24 h

1, TBAF, rt, 5 h

2, TFA, 10 min

Py / DMAPrt, 24 h

DMAP, Hunig's basepy, DMF, rt, o/n

TPS-Cl(2 equiv)

R2

R1R2NH

Y Y

Y

Y

S

Pri

Pri

Pri

O

O

-OCH3

DCM

OCH3

An 1009

N

NO

O

O S

OSi

OMe

O

O

O

CH3

MAS NMR of 2’-OMeU-4-sulfonate MMT Resin

6,8-Disubstituted Adenosine Library

12 plates

1148 compds

NN

NN

NH2

O

OHHO

HO

BrN

N

NN

NH2

O

OHHO

HO

R1

NN

NN

NHAc

O

OAcAcO

HO

R1

R1X

Heck or

Suzuki

1) TBDMSCl

2) Ac2O3) TBAF

ClN

N

NN

N

O

OAcAcO

R1

O

AcR2

NN

NN

HN

O

OHHO

R1

HO

R2

R2-OH

Ph3P/DEAD

Mitsunobu

1) NH3 deprotection

2) TFA cleavage

8-Amide Adenosine Library

12 plates

1152 compds

N

N

NN

NH2

O

OHHO

HO

MeOCON

N

NN

NHAc

O

OAcAcO

HO

MeOCO

N

N

NN

NHAc

O

OAcAcO

MeOCO

O

1) TBDMSCl

2) Ac2O3) TBAF

Cl

N

N

NN

N

O

OAcAcO

MeOCO

O

AcR1

N

N

NN

HN

O

OHHO

HO

R1

TFA

cleavage

R1-OH

Ph3P/DEAD

Mitsunobu

HNR2R3

N

O

R3 R2

2-Amino Adenosine Library

48 X 48, 24 plates

2304 compounds

N N

NN

Cl

O

OHHO

HO

ClN NH

NN

O

O

OHHO

HONH2

I4 steps

N N

NN

N

O

OHHO

ON

R1 R2

R3

R4

1% TFA cleavage or

hexafluoroisopropane

R1R2NH

THF, Toluene

or NMR

rt - 60 ºC

R3R4NH

THF, Toluene

or NMR

80 - 100 ºC

N N

NN

N

O

OHHO

HON

R1 R2

R3

R4

N N

NN

Cl

O

OHHO

OI

Exo-cyclic Triazine Nucleoside Library

N3O

OHHO

HONH2O

OTBDMSTBDMSO

O

NHO

OTBDMSTBDMSO

O

N

N N

Cl Cl

NHO

OHHO

HO

N

N N

HN N

1) MMT-Cl Resine

2) TBDMS-Cl

N

N

N

Cl Cl

Cl

R1NH2 (primary)

Hunig's base

rt 2-5 h

R2NH2 or R

2NHR

heat 5h

Hunig's base

- 20 °C

1) TBAF

2) TFA

R1

R2

R

3) PMe3

48 X 24, 12 plates

1152 compounds

a. 4 - M e t h o x y-t r i t y l c h l o r i d e-P S-r e s i n

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0 1.4

4

6

8

10

12

14

16

18

20

22

24 25.0

cm-1

%T

Cl

OCH3

c. (4) 1 - A m i n o - s u g a r o n r e s i n

4000.0 3000 2000 1500 1000 450.0 1.4

4

6

8

10

12

13.5

cm-1

%T

b . (3) 1-A z i d o-s u g a r o n r e s i n

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0 0.24 0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5 5.83

cm-1

%T

O N3MMTO

OTBDMSTBDMSO

O NH2

MMTO

OTBDMSTBDMSO

FT-IF Spectral Comparison of Different Resins

Clitocine Mimic Exocyclic Amino Nucleoside Library

82 compds

O NH

HO

N

N

N

OHHO

R4

R5

O NHMMTO

N

N

Cl

OTBDMSTBDMSO

HNR4

R5

NO2

NO2

O NH2

MMTO

OTBDMSTBDMSO

Cl

N

N

Cl

NO2

Hunig's base

TBAF TFA

N1- and N2-Methylpyrazolopyrimidine Nucleoside Libraries

96 compds

87.5% successful rate

96 compds

97.9%

successful

rate

N

N N

N

H3CS

OO

HO OH

CH3

O

N

N N

N

NH

OO

HO OH

CH3

O

1.0 M Amines in NMP

80 ºC 24 hrs

R

N

N N

N

NH

O

O

HO OH

CH3

HO

R

30% HFIP in DCE

50 ºC 24 hrs

N

N N

N

H3CS

OO

HO

HO OH

CH3

MMT resin- Cl/

2,6-Lutidine/DMF

N

N N

N

H S

O

O

HO

HO OH

CH3

MeI/DIPEA

N

N N

N

H O

OO

BzO

BzO OBz

CH3

1) P2S5/Pyridine

2) NaOMe/MeOH

N

NN

N

NH

OO

HO OH

HO

R2

CH3

N1

N2

Attached at Other Positions on Base

NO

OCH3AcO

N

O

OTPS

AcO

MMTCl

R3

N

NO

OCH3HO

N

OHO

Pyr. / DMAP / TEA,rt, 24 h

Pyr. / DMAPrt, 24 h

TPS-Cl

(2 equiv)

NO

OCH3AcO

NH

O

O

AcO

R4

HNR3

R4 1) NH3/MeOH

2) TFA

HO

HO

O

Attached at 3’-Position

NO

OCH3

N

O

OTPS

TBDMSO

MMTCl

R3

N

NO

OCH3

N

OHO

Pyr. / DMAP / TEA,rt, 24 h

Pyr. / DMAPrt, 24 h

TPS-Cl(2 equiv)N

O

OCH3

NH

O

O

TBDMSO

R4

HNR3

R4 1) TBAF

2) TFA

OHO

HO

Steps

NH

N

N

O

NH2N

O

OAcAcO

AcO

R1

NH

N

N

O

NH2N

O

OAcOAc

AcO

Br

N

N

N

N

NH2N

O

OHHO

HO

R1

N

N

N

NH

N

O

OHHO

HO

R1 MMT

R2 R3N

R2 R3

N

N

N

Cl

NH

N

O

OAcAcO

AcO

R1 MMT

Suzuki Still Heck

reactions

POCl3

PhNMe2

Et4NCl

Cl-MMT

R2R3NH

NMP

60 oC, 6h

80 o

C

24 h

CH3NH2

CH3OH

50 oC, 24h

HFIP/DCE

50 oC, 24h

Attached at N2-Position on Base

5 plates 480 compds

Protection &

attachment

H2N H2NH2N OHH2N

H2N O H2NO

H2N

H2N H2NS

H2N

N

H2N

O

H2N

H2N

H2N

H2N NH2N

H2N

H2N

NONH2

H2N

H2NCF3

H2N

NNH2N

O

OH2NN

H2N

N

H2N

NH2

H2N

O

NH2NN

O

H2N

H2N

NH2

NH2O NH2

O NH2

NO NH2

NNH2

O

H2N

N NH2

H2N

HO

N NH2

OH2N

SH2N

H2N

CF3H2N

NH2

NNH2

H2N

H2N

H2N

ONH2

O

HO NH2

OH

H2N

NH2

H

H

NH2H

H

H2N OH

H2NO

ONH2

NH2N

NNH2

NH2

HONH2

N NH2

NH2

O

NH2

H2N

H2N

O NH2

H2N

NH2

H2N

H2N

H2NO

H2N

FNH2

H2N

H2NH2N

H2N

H2N

NH2

NH2

F

NH2

F

NH2

FNH2

F

H2NO

H2N

H2NOH

HO

HO

H2N OHH2N

H2N

HO

NH2

NH2

OHNH2

H2N

H2N

H2N N

H2N H2N

H2N OEtH2NOMe

H2N H2N H2N SH2N H2N

NH2N

O

H2N

N

Ph

H2N H2NH2N NH2N

H2NH2N

N O

H2N H2N

H2NPh

H2NN NH2N H2N

H

H

PhNH

H2N

Ph

H2N O

O

H2N H2N

H

H

NO2

N

NHH2N

NH2N

NH2NH2NOH2N

NH2N

N

O

H2N

H2N

OH2N

N OH2N

NH2N H2N

OH

O

H2NPh

HO

H2N

H2N

N

PhOH2N SH2N

H2N

OOH

H2NO

H2N

NH2N

H2N

O

H2N N

NH2

H2N

Ph

H2N H2N

1 2 3

49 50

4 5 6 7 8

9

56

1011 12 13

14 15

16 1718

19

20 21

22 23 24 25

27

26

2928 30

51

52

31

33

3435

36 37

54

38 39

4041

42

55

43 4445

46 47

H2N

32

48

HN H

N HN OHHN

HN NH

OH

O

OHN

PhNHN

NH

OH

N

N

HN NHN

HO

NHN N

O

HN N

N

N

HN

HNHN

HN S

NH

HN

NH

HN HN O NH

PhNHN

1 2 3

5 67

4

8

9

1011

12 13 14

15 16 17

18

1920

21 232422

Some Representative Building Blocks Utilized

Semi-Automated Synthesizer

VanguardVanguard

96-Well

Parallel

Solid-Phase

Synthesizer

Hardware Solutions

TH Parallel Characterization System

Alliance HT LC/MS

MassLynx Control Alliance HT 2790PDA 996

ZQ Mass Detector

Library

C-3

LC-MS Results of A Representative Plate

NO

N

N

OHOH

N

NH

OH

O

NH

O

A1, M.W. 520.55

LC-MS of a Library Member

NO

OCH3HO

HO

N

O

HN

N

C18H30N4O5

Mol. Wt.: 382.45

383

(M + 1)+

(1)

382

0.42

100%

LC-MS of a Library Member

NO

OCH3HO

HO

N

O

HN

N

C19H34N4O5

Mol. Wt.: 398.50

399

(M + 1)+

398.25

0.44

100%

LC-MS of a Library Member

Library Quality

Plate #

Successful

Rate*

(%)

Compds (%)

with 80-100%

Purity

Plate #

Successful

Rate*

(%)

Compds (%)

With 80-100%

Purity

1 33.3 16.6 11 91.7 81.2

2 41.2 16.0 12 93.8 82.3

3 66.3 56.3 13 94.8 69.8

4 75.0 45.8 14 94.8 89.6

5 72.0 57.0 15 95.8 62.5

6 76.0 60.0 16 95.8 84.3

7 87.5 71.9 17 96.9 74.0

8 85.0 85.0 18 97.9 92.7

9 87.6 72.0 19 97.9 93.7

10 92.7 71.0 20 100 91.7

* percentage of compds with >60% purity is defined as successful rate

HT Parallel Purification System

Biotage Parallex Flex HPLC

Software Solutions

Afferent TeamWorks– Enumerate library, generate compd and impurity profiles

MassLynx Control Software– Generate sample list as Excel file (ID, MW)

OpenLynx Software

– Automatic spectral (MS, UV) and chromatographic data

processing

– ID results in color-coded fashion & 96-well format

Activity Base/Customized Program

– Cherry-Pick compds: registration vs. repurification

– Enter analytical data into database

Conclusions

Developed versatile parallel solid-phase combinatorial technology for the synthesis of diverse nucleoside libraries

Various positions on nucleoside scaffolds can be attached onto solid supports

Diverse purine-, pyrimidine, pyrazolopyrimidine and triazine-based nucleoside libraries were synthesized in high quality and speed

All library members were characterized by HT

LC-MS. Some were characterized by NMR

Applicable to other nucleoside derivatives

Acknowledgements

Chemistry Team:Frank Rong

Qazi Habib

Roberto R. Amador

Kanda Ramasamy

Varaprasad Chamakura

Esmir Gunic

Yung-hy Koh

Stephanie Shaw

Yili Ding

Jean-Luc Girardet

Huanming Chen

Jeffrey W. Abt

Weijian Zhang

Zhi Hong

Stanley Lang

Inventory & HTS:Maja Stojiljkovic

Vesna Stoisavljevic

David Hyndman

Paul Diaz

Analytical:Jingfan Huang

David Y. Li

Micheal Landsman

Xiaogang Han

Teresa Cain

CAAD

Biochemistry

Biology

Virology

Oncology